焦磷酸测序法检测HBV前C区／基本核心启动子突变的临床应用

Pyrosequencing of pre-C/BCP mutation in hepatitis B

目的建立一种新的基于焦磷酸测序技术的HBV前C区／基本核心启动子（pre-C／BCP）突变的检测方法，并验证该方法的准确性、重复性、可靠性及进行初步临床检测。方法通过对基因库中100个HBV基因序列的比对分析，设计出专用的pre-C／BCP焦磷酸测序的PCR扩增引物及测序引物，并以标准质粒作为标准品建立其相应的检测方法。通过对标准质粒及PCR扩增产物、经Sanger测序的HBV血清及基因芯片检测结果分析，以验证本检测方法检测的标本的特异度及本方法的检测可靠性。最后对60例HBV临床血清标本进行了初步的检测。结果本研究成功地建立了pre-C／BCP突变热点的焦磷酸测序方法。其检测结果与pre-C／BCP质粒，Sanger测序法的检测结果符合率达100％（Kappa=1.0），基因芯片检测结果的诊断符合率为91．7％。同一批血清标本的重复率达97．8％，阳性PCR产物测序的重复率100％。充分证明了本方法的准确性、重复性、可靠性。初步批量化检测60份临床标本结果表明，本方法能一次批量检出包括pre-C／BCP的单一、多位点的突变，并发现了一例少见的BCP T1758C突变。结论本实验所建立的pre-C／BCP突变热点的焦磷酸测序方法是一种可一次性检测HBV pre-C／BCP多位点突变的高通量的检测方法。

Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/ BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 clinical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/ BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.